原汁原味的慢病毒转染与感染protocol

Lentivirustransfection and infections

Packaging cell line:

293FT

Growth medium:

DMEM containing 10% HI FBS, 1%P/S, 0.5mg/ml G418,4 mM L-Glutamine,0.1 mM MEM Non-Essential Amino Acids,1 mM MEM sodium pyruvate

Lentiviral medium:

DMEM containing 10% HI FBS, without antibiotics,4 mML-Glutamine, 0.1 mM MEM Non-Essential Amino Acids,1 mM MEM sodium pyruvate

Targetcell medium:

DMEM or other medium containing 10% HIFBS, without antibiotics.

Protocol

Culturing 293FT cells

Quickly thaw the frozen 293FT cells, and immediately after thawing, transfer the cells to a 15 ml tube containing 10 ml PBS,and then pellet the cells. Resuspend the cells in growth medium, and incubate cells at 37°C in a humidified 5% CO₂ incubator.

Transfection and infections

1. On Day 1, prepare DNA-Lipofectamine? 2000 complexes foreach sample.

a.In a sterile 5 ml tube, dilute 3 μg pLP1, 3 μg pLP2, 3 μg pLP/VSVG, and 3 μg of pLenti expression plasmid DNA (12μg total) in 1.5 ml of DMEM (noserum, no P/S). Mix gently.

b.In a separate sterile 5 ml tube, dilute 36 μl Lipofectamine? 2000 (mix gently before use) in 1.5 ml of DMEM (no serum, no P/S). Mix gently and incubate for 5 minutes at room temperature.

c.After incubation, combine the diluted DNA (Step a) with the diluted Lipofectamine? 2000 (Step b). Mix gently.

d.Incubate for 20 minutes at room temperature to allow the DNA Lipofectamine ? 2000 complexes to form. The solution may appear cloudy, but this will not impact the transfection.

e.While DNA-lipid complexes are forming, trypsinize and count the 293FT cells.Resuspend the cells at a density of 1.2x 10⁶ cells/ml in lentiviral medium.

f.Add the DNA-Lipofectamine? 2000 complexes (Step 1d) to a 10 cm tissue cultureplate containing 5 ml of lentiviral medium.

g. Add 5 ml of the 293FT cell suspension from Step 2 (6 x 10⁶ totalcells) to the plate containing media and DNA-Lipofectamine? 2000 complexes (Step 3).

Mix gently by rocking the plate back and forth. Incubate cells overnight at 37°Cin a humidified 5% CO₂ incubator.

2.The next day (Day 2),remove and discard the medium containing the DNA Lipofectamine ? 2000 complexes and replace with 10 ml lentiviral medium.

Incubate cells for 48 hours at 37°C in a humidified 5% CO₂ incubator.

Note:Expression of the VSV G glyco protein causes293FT cells to fuse, resulting in the appearance of large, multinucleated cells known as syncytia. This morphological changeis normal and does not affect production of the lentivirus.

3. Day 3, set up the targetcell line in target cell medium to 60 mm plate so that they will be 30% confluent on the next day.

4. Posttransfection (Day 4),

a. harvestvirus-containing supernatants. Use a 10 ml syringe to remove the medium from the 293FT cell lines, and filter the viral supernatants through a 0.45 μm filter in 15 ml sterile tube. Replace the medium removed from the packaging cells with 10 ml lentiviral medium.

b. infect target cells: 1 volume of lentiviral medium (2ml) and 1 volume offiltered virus-containing supernatants(2ml), with polybrene 4 μg/ml.

c.Pipet the retaining viral supernatants into 1.5 ml sterile tube in 1 ml aliquots. Store viral stocks at -80°C. When stored properly, viral stocks ofan appropriate titer should be suitable for use for up to one year. When using the frozen viral stocks, thaw frozen stocksat room temperature, rather than at 37°C, since lentivirus is temperature-sensitive.

5. Day 5 or 6, second round of infection: repeat the infection as described on day 4. And throw the 293FT cells.

6.Day 6 or 7, split target cells to 100 mm plate with fresh target growth medium.

7. On the Day 7or 8, select cells using drug.